Gamma H2AX antibody and the DNA damage reagent set: read foci, not protein

Designing a damage experiment

1. Decide lesion, signal or route. An oxidised base, a signalling mark spreading from a break, and a repair factor loading are three different observations. Naming which you need selects the reagent and also the readout, because only some of them are meaningful as foci counts.
2. Count foci rather than measure intensity. The signalling mark spreads over a large chromatin domain around each break, so the informative measurement is the number of discrete foci per nucleus. Whole nucleus intensity mixes that with background and with cycle stage.
3. Control for replication. Cells in S phase carry damage marks and repair factor foci as a normal part of replication. Without a cycle marker in the same image, an apparent increase in damage can simply be more cells replicating.
4. Use a defined dose and a time course. Foci appear within minutes and resolve over hours, and the resolution curve is the informative part. A single late timepoint reports repair capacity as if it were damage.
5. Include a positive damage control on every slide. A known dose of a damaging agent on the same slide calibrates the stain and shows the reagent worked. Damage experiments without one are difficult to interpret across days.

Repair route is inferred, not observed

Loading of a recombinase or a resection factor indicates that a route is being attempted; it does not prove the break was repaired that way, and several factors participate in more than one route. Conclusions about route need more than one marker and preferably a genetic manipulation.

State inference as inference. A figure showing more recombinase foci supports a claim about pathway usage; it does not by itself demonstrate it.

Dose, time and cycle are the three axes

Every damage experiment varies along dose, time after damage and position in the cycle, and a result that fixes two of them and varies the third is interpretable. Fixing all three and comparing conditions is where most confusion enters.

Record all three with every image set. It is the metadata that makes a damage figure re-analysable rather than merely illustrative.

primary antibody western blot practice

A primary antibody western blot is set up by titrating the antibody against a positive control lysate rather than by taking the datasheet dilution, since membrane, blocking buffer and detection system all shift the optimum. Incubation overnight in the cold gives lower background than an hour at room temperature for most clones. The exposure, the blocking agent and the dilution belong in the figure legend.

western blot primary antibody choices

Choosing a western blot primary antibody is a question about the denatured epitope: a clone raised against a native surface may not see a reduced, boiled protein at all, which is why the datasheet's validated applications matter more than its affinity. Where a phospho signal is wanted, inhibitors in the lysis buffer and a total protein blot on the same membrane are part of the choice.

antibodies for western blotting, and how a set is stocked

A laboratory stocking antibodies for western blotting keeps a small set of secondaries against the hosts it uses, a loading reference suited to its fractions, and a molecular weight ladder its detection system can see. The primary list follows the projects. Aliquoting on arrival and recording the lot are what stop a good antibody becoming an unreproducible one.

An nbs1 antibody and the repair complex it belongs to

NBS1 works in a complex with MRE11 and RAD50, so an nbs1 antibody is read as recruitment to damage with its partners rather than as a level, and the experiment is damage followed by focus formation on a timescale. Its own abundance changes little. A patient derived line with reduced protein is the negative control the field uses.

An mre11 antibody and the nuclease in that complex

MRE11 is the nuclease of the same complex, so an mre11 antibody is bought with the others for a co-localisation or a co-immunoprecipitation rather than alone. It is also the target of a well known inhibitor, which gives a functional control. Because the three subunits depend on each other for stability, knocking one down lowers the others, which is the expected result.

An mdc1 antibody and the mediator that amplifies the signal

MDC1 binds the phosphorylated histone this page's own antigen detects and then recruits the rest of the response, so an mdc1 antibody is read in the same image as that mark and the readout is focus overlap. It is a very large protein, which makes transfer the usual failure on a blot, and the foci resolve with time, so the imaging point is part of the method.

Common questions

Does a damage marker measure the number of breaks?

Approximately, at early timepoints and low doses, when foci are resolvable. At high dose foci merge and the count saturates, and at late timepoints the count reflects unrepaired damage rather than initial damage.

Why do untreated cells show foci with a RAD51 antibody or gamma H2AX antibody?

Replication generates damage signals as a normal part of copying the genome. Staining a cycle marker alongside, and gating on non replicating cells, separates that from induced damage.

Foci or western blot?

Foci for spatial information and per cell distribution, which is what most damage questions need. A blot averages across the population and can miss a large response in a small subpopulation entirely.

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